Hop Proanthocyanidins for the Fining of Beer

Home , Isinglass

1 Hop proanthocyanidins for the fining of beer

3 R. S.T. Linforth*a, K. Westwoodb, A. Somanic, N. Dohertyc, D.J. Cookc

6 aFood Sciences, School of Biosciences, Sutton Bonington Campus, University of Nottingham.

7 LE12 5RD. UK.

8 bBarth Innovations Ltd, Hop Pocket Lane, Paddock Wood, Kent, TN12 6DQ, United Kingdom

9 cBrewing Science Department, School of Biosciences, Sutton Bonington Campus, University of

10 Nottingham.

11 LE12 5RD. UK.

15 *Corresponding author: [email protected]

19 Abstract

20 Fining agents are used in the clarification of beers; they help to reduce the time required to

21 sediment suspended yeast cells and ensure the clarity and colloidal stability of beer.

22 Following an adventitious observation during dry-hopping experiments, we identified a

23 fining activity associated with Saaz hops. Extracts of hop cones were subsequently shown to

24 have the capacity to flocculate yeast and result in their sedimentation. This activity has since

25 been identified in extracts of many different hop varieties and, significantly in spent hops,

26 the co-product resulting from commercial extraction of hops with either CO2 or ethanol.

27 Here we illustrate the activity of the novel finings extracted from spent hops following CO2

28 extraction of Galena hops. The sediments formed on fining were compact, relative to those

29 obtained when commercial isinglass was used to fine the same beers. The hop extracts were

30 also effective in reducing 90° haze in beers under conditions designed to mimic both cask

31 ale (12°C) and lager (4°C) type applications.

32 The compounds responsible for the fining activity appear to be large (30 to 100kDa, or

33 more) polyphenols. Analysis of the polyphenols using colourimetric tests, indicated the

34 presence of proanthocyanidins. On acidic hydrolysis these generated cyanidin, which would

35 be derived from a polymer composed of catechin and epicatechin subunits. The presence of

36 these materials in spent hops offers the possibility to develop commercial products, with

37 desirable fining properties, from an existing co-product stream. Furthermore, the finings are

38 derived from a traditional ingredient of the brewing process.

41 Key words. Fining agent, hop polyphenolics, proanthocyanidin, brewing, colloidal stability.

43 1. Introduction

44 The vast majority of beers consumed worldwide are intended to be served clear, bright and

45 free from visible haze. It is important that clarity is achieved in fresh beer and that this is

46 retained through the required shelf-life, such that beers are delivered to the consumer in

47 optimal condition. Haze can be considered as the ‘absence of clarity’ and is caused by the

48 presence of small insoluble particles, typically in the μm size range, which scatter light,

49 leading to the perception of haziness (1). There are several different sources of haze in

50 beers, ranging from sporadic negative factors such as microbial infection, through

51 precipitates of relatively insoluble salts such as calcium oxalate, to the presence of colloidal

52 materials (e.g. proteins, carbohydrate polymers) which are only sparingly soluble in the beer

53 matrix and therefore have a tendency to form insoluble aggregates of material, leading to

54 colloidal instability (2). Of particular relevance are the complexes formed between

55 polyphenols and so-called haze-sensitive proteins, which are responsible for chill-haze in

56 beer (3; 4; 5) (haze material which comes out of solution when beers are refrigerated but

57 which dissolves when returned to 20°C). Ensuring the colloidal stability of beer involves

58 control of factors across the brewing process from raw materials selection, through

59 brewhouse processing and into finished beer (6). The maturation period, post-fermentation,

60 is particularly significant in this regard. Green beers contain residual yeast cells in

61 suspension, a factor which in itself can lead to haziness of beers if steps are not taken to

62 remove them. Traditionally, the clarity of lager beers has been ensured by cold-conditioning

63 them for periods of several weeks, during which the insoluble materials settle out to form

64 ‘tank bottoms’, leaving behind bright beer. In the modern day industry it is not desirable to

65 incur the costs of chilling and storing large quantities of beer, hence rapid maturation

66 processes have been developed to ensure colloidal stability of beers over much shorter

67 time-frames. These usually involve the use of process-aids designed to selectively remove

68 haze materials, or their precursors. Examples would be the use of PVPP to lower the

69 polyphenolic precursors of haze, or of tannic acids or silica gels to remove portions of the

70 haze-sensitive proteins in beer (7). Physical separation processes such as centrifugation and

71 filtration are also used to remove particulates, however, a combination of approaches is

72 often required in order to ensure that the loading of solids in rough beer does not lead to

73 blinding of filters, or shortening of effective filtration run times.

74 Fining agents are used to accelerate the rate of separation of suspended particles from

75 beers and in general work by cross-linking haze particles to generate larger aggregates of

76 material which settle out more rapidly to the bottom of a vessel. Finings have traditionally

77 been used most prevalently in the production of cask ales in the UK. Since cask ales contain

78 live yeast in contact with the product and undergo secondary fermentation in the trade, it is

79 necessary to clarify such beers by the addition of fining materials, such that yeast settles out

80 efficiently on completion of the secondary fermentation, to form a compact sediment.

81 Furthermore, the use of finings to treat brewery-conditioned beers has become more

82 widespread, as part of the overall strategy of ensuring colloidal stability using shorter

83 process times (8).

84 The most widely encountered finings material in brewing is isinglass, a purified protein

85 preparation extracted with dilute acid from the swim-bladders of certain species of tropical

86 or sub-tropical fish. The active ingredient is almost pure collagen (9). Isinglass acts by cross-

87 linking suspended yeast cells, via a charge-interaction, leading to their aggregation and

88 subsequent sedimentation. Isinglass carries a net positive charge at beer pH’s, facilitating its

89 interaction with the negatively charged surface of yeast cells (8; 10). The use of isinglass is

90 well established in certain applications and regions of the world, mainly because isinglass

91 combines several features attractive to brewers. In addition to the flocculation of yeast

92 cells, isinglass is also active against chill-haze (8), forms sediments which are compact

93 (leading to minimal beer losses and easier run-off of beers from above the sediment),

94 improves subsequent filterability of beers and has been noted to improve beer foam (8),

95 most likely due to the removal of foam negative lipid materials. However, one aspect which

96 limits the usage of isinglass is the fact that it originates from fish swim bladders, meaning

97 that products manufactured using isinglass are not suitable for vegans and are not

98 considered kosher. At one time it was proposed that residues of isinglass in beer might pose

99 a threat to fish allergy sufferers and that products would need to be labelled accordingly

100 (11; 12); however, this requirement did not become law in the EU because it was possible to

101 prove that residual isinglass levels in treated beers were extremely low, hence did not pose

102 a threat (12). Due to the aforementioned concerns, researchers have attempted to identify

103 alternative fining materials with which to treat beers and wines. These have included

104 evaluation of avian collagen and pea protein extract (10), the use of plant pectins (13), or of

105 bovine collagen (14). To date none of these approaches have been exploited commercially,

106 probably because none of the materials individually match the performance of isinglass in

107 all of its beneficial features. Thus isinglass remains the only finings material in widespread

108 brewing usage. In spite of this, there are other aspects to the use of isinglass which might be

109 improved upon when developing novel fining agents; isinglass is not an easy material to

110 disperse and mix into water. The UK is the only region with a significant market in wet

111 isinglass products whereas the remainder of the world principally uses dry isinglass powder

112 which must first be dispersed in water to the appropriate strength, prior to dosing into the

113 process. Once these solutions have been prepared they have a limited shelf-life and need to

114 be stored refrigerated (4-10°C) to retain activity; at higher temperatures collagen rapidly

115 denatures to inactive gelatine (1).

116 In this paper we describe the characterisation of a novel fining material which has the

117 potential to compete with isinglass in brewing applications. The novel finings is sourced

118 from hops, and can therefore be promoted as a natural ingredient of the brewing process;

119 although with conventional usage of hops in brewing the compounds believed to confer

120 fining activity would not typically persist into the product. Furthermore, the active material

121 is shown to be extractable from spent hops, the co-product generated through the

122 extraction of hop resins using liquid CO2 or ethanol.

123 2. Materials and methods

124 Chemicals

125 Analytical grade acetic acid, ferric ammonium sulfate, butanol, and High Performance Liquid

126 Chromatography (HPLC) grade acetone, acetonitrile, and ethyl acetate were purchased from

127 Fisher Scientific (Loughborough, UK).

128 Materials

129 Hops (variety Galena) that had previously been extracted by CO2 were provided by Barth

130 Innovations Ltd (Paddock Wood, Kent, UK). Liquid Isinglass, AllKleer A, was purchased from

131 Murphy and Sons Ltd (Nottingham, UK). Dry yeast (Youngs, Bilston, Uk) was purchased from

132 the Hop Shop (Plymouth, UK).

133 Preparation of hop extracts

134 Hops were extracted using either water, or 70% acetone in water. Aqueous extracts were

135 prepared by mixing hops with reverse osmosis purified water (15mL/g hop) on a rollerboard

136 for 30min at room temperature. The extract was then crudely filtered using muslin cloth,

137 centrifuged at 7500rpm, 4°C, for 20min (Beckman, J2-21M, High Wycombe, UK) and the

138 supernatant sequentially filtered (Whatman No.1, 3, 5, 602, purchased from Fisher

139 Scientific; 0.45µm hydrophilic syringe filters, Sartorius Stedim, Germany) and stored at -18°C

140 prior to use.

141 Extraction into 70% acetone in water involved mixing the hops with solvent (15mL/g hop) on

142 a rollerboard for 2h at room temperature. The extract was then filtered using Whatman

143 No.1 filter paper and the acetone removed by rotary evaporation (Buchi, Rotavapor II,

144 Labortechnik AG, Flavil, Switzerland). The aqueous solution was then adjusted to pH 4

145 (InoLab pH level 1, Wissenschaftlich Technische Workstätte, Weilheim, Germany) using HCl

146 and partitioned against an equal volume of ethyl acetate. The aqueous phase extract was

147 retained, rotary evaporated to remove any residual ethyl acetate and stored at -18°C prior

148 to use.

149 Preparation of green beer

150 Youngs Economy Pilsner kits (Young’s Home Brew, Bilston, UK) were purchased from the

151 Hop Shop (Plymouth, UK) and fermented following the instructions on the label for 96h at

152 22°C. The green beer was then syphoned into a separate container to leave behind yeast

153 that had already sedimented.

154 Sedimentation studies

155 Clarity of green beer was determined at OD 600nm using a UV-Vis spectrophotometer

156 (Jenway, 6315, Stone, UK). Sedimentation volumes, were determined by mixing the hop

157 extract with green beer in Imhoff cones (1L, VWR, Lutterworth, UK) and leaving them for

158 24h at 4°C. The beer was partially de-gassed by stirring before the application of the hop

159 extract, or, Isinglass.

160 Size filtration studies

161 The extracts were sequentially size fractionated using reconstituted cellulose, molecular

162 weight cut off filters, at 100, 50, 30, 10, and 3kDa, Amicon, Ultra-15 centrifugal filter units

163 (Millipore, Watford, UK).

164 HPLC fractionation

165 Hop extract (aqueous, 1mL) was injected onto a 250 x 4.6mm cyano (CN) column

166 (Phenomenex, Macclesfield, UK) and eluted isocractically with a binary solvent mixture of

167 30% acetonitrile and 70% 0.1% acetic acid at 0.6mL/min. The eluent was collected as

168 separate 0.9mL fractions. The activity of each fraction was determined by the addition of

169 100µl of each fraction to 10mL of green beer and observing the sedimentation of yeast.

170

171

172 Acidic Butanol hydrolysis of the active hop extract

173 The active fraction obtained from HPLC fractionation of the hop extract (200µl) was added

174 to 3mL of 5%HCl in butanol and 0.1mL of 2% ferric ammonium sulfate in 2N HCl. The

175 mixture was subsequently heated to 100°C for 20min.

176 Mass spectral analysis of the acidic butanol hydrolysate

177 The butanol-HCl hydrolysed extract was introduced into the electrospray source of a

178 Micromass LCZ mass spectrometer (Manchester, UK) operated in positive ion mode at

179 10µL/min using a syringe pump (Harvard Apparatus, Edenbridge, UK). The source

180 temperature was 300°C, the desolvation gas was nitrogen at 350L/h with a cone voltage of

181 60V. Mass spectra were recorded over the mass range m/z 250-350 with a scan rate of

182 0.5Hz.

183 Haze analysis

184 Haze in beer was evaluated as total haze. Beer treated with finings was allowed to clear and

185 was then transferred into 50mm dia. glass cuvettes and the amount of light scattering at a

186 measuring angle of 90° determined using a turbidimeter (Norit Haffmans Vos Rota 90/25,

187 Germany).

188 2 protocols were adopted, one at 12°C where the fining agents were applied and the haze

189 measured (at 12°C) after 72h, with no further treatment. In the second, fining agents were

190 added at 4°C and the haze was determined (at 4°C) before and during filtration through

191 sequential 11, 3 and 0.45µm filters.

192 3. Results

193 Preliminary studies found that aqueous extracts of whole cone hop samples (variety Saaz)

194 could induce the flocculation and sedimentation of yeast cells in green beer. Subsequently,

195 the same activity was observed for extracts prepared from spent hops, a by-product

196 resulting from the commercial extraction of hops with liquid CO2. These latter extracts were

197 prepared and used in further studies.

198 Sedimentation studies

199 The addition of the hop extract to green beer caused yeast to flocculate and sediment. This

200 resulted in a decrease in the OD 600nm of the beer (Figure 1). As little as 5mL/L of the hop

201 extract (equivalent to 0.33g of original hop material/L) was sufficient to induce a reduction

202 in the OD 600nm. Dose rates beyond 20mL/L had little further impact on OD 600nm

203 reduction. This level (20mL/L) was thus identified as the optimum dosage for the hop

204 extract in this beer for use in sedimentation trials.

205 Sediment volumes produced by the hop extract were compared with the corresponding

206 sediments resulting from the use of commercial Isinglass. Both solutions were added to

207 green beer in proportion to their optimum dose (20mL/L as determined by OD 600nm

208 studies). After 24h, all beers had clarified and the yeast sedimented. The sediment volumes

209 were more compact at 22°C, for both the Isinglass and the hop extract, than at 4°C (Figure

210 2). The hop extract produced smaller sediment volumes than Isinglass, at comparable dose

211 rates, at both temperatures. Addition of larger volumes of isinglass and the hop extract both

212 resulted in larger aggregates of yeast and hence sediment volumes.

213 In addition to the flocculation and sedimentation of yeast in green beer, it was found that

214 the hop extract could be used to sediment suspensions (1% w/v) of hydrated (75min) dry

215 yeast (data not shown). This was used as an assay to determine the presence or absence of

216 flocculation activity in further studies.

217 Characterisation of the active compound

218 Aqueous and 70% acetone (aq) hop extracts were size-fractionated using molecular weight

219 cut off filters mounted in centrifuge tubes. These separate the filtrates from the retentate,

220 with molecules passing through the filters or not, depending on their molecular weight. A

221 series of filters were used sequentially to profile the size range of compounds in the extract;

222 the presence of active compounds in the various fractions generated was detected by

223 observing the flocculation and sedimentation of re-hydrated yeast. The smaller the volume

224 of an extract required to flocculate the re-suspended yeast the greater the activity.

225 The activity of the aqueous extract was not retained by 100, 50 or 30kDa filters, but, was

226 detected in the 10kDa retentate. No activity passed through this filter into subsequent

227 fractions. The active component in the aqueous extracts thus appears to be in the molecular

228 weight range from 10 to 30kDa.

229 The acetone extract produced a range of active fractions. The 100kDa retentate induced

230 yeast flocculation with 4mL of the retentate, indicating the presence of higher molecular

231 weight material than the aqueous extract. The 50-100kDa and 30-50kDa fractions both

232 required 8mL of the retentate to induce the flocculation response, whereas the 10-30kDa

233 fraction required 15mL. No activity was detected in the 3-10kDa fraction, but, additional

234 activity was detected in the <3kDa fraction, equivalent to that observed for the 100kDa

235 retentate. The acetone extract appeared to mostly contain higher molecular weight

236 polymers, with less and less activity in lower molecular weight fractions. The activity

237 observed in the <3kDa fraction could indicate that there were 2 active components in the

238 extract, one in the high molecular weight range and one at low molecular weights.

239 Alternatively, the <3kDa activity could be due to fragments of the higher molecular weight

240 material from the hop itself, or, formed during extraction.

241 An extract was also fractionated by HPLC using a cyano column. The active compound

242 eluted in a fraction with a retention time of between 7.5 to 9min. An active extract from

243 cyano fractionation was hydrolysed in acidic butanol, which is a test for the presence of

244 proanthocyanidins (15). The solution turned red, indicative of the presence of

245 proanthocyanidins in the active fraction. The visible spectrum of the solution showed a

246 maximum absorption at 552nm (Figure 3) typical of anthocyanidin formation from

247 proanthocyanidins during hydrolysis (15).

248 Visible spectra can help with the identification of anthocyanidins. There are however a

249 number of different anthocyanidins that could be produced by the breakdown of

250 proanthocyanidins and the observed spectra can also be affected by the solvent, or the pH

251 of the solvent, in which the spectra are recorded. To help with identification of the

252 anthocyanidin the extract hydrolysed with butanol/HCl was analysed by direct infusion mass

253 spectrometry. The resulting spectrum showed a major ion at m/z 287 (Figure 4), which is

254 consistent with the presence of either cyanidin or robinetinidin. Cyanidin and robinetinidin

255 have visible absorption maxima of 535 and 525nm respectively (16) which differ from those

256 observed, but, this may be a solvent-related difference. Robinetinidin can be produced by

257 the hydrolysis of quebracho tannin. Proanthocyanidins producing cyanidin are more

258 common (17), and derive from proanthocyanidins containing catechin and epicatechin as

259 the polymer sub units (15). Catechin and epicatechin are optical isomers of one another.

260 Based on these results, the active compound in our hop extracts appears to be a large

261 polymeric proanthocyanidin, comprised of catechin and epicatechin subunits.

262

263

264

265 Activity of hop extracts against haze in beer

266 Two protocols were used for the beer haze studies, designed to mimic the two major

267 applications of isinglass finings. The first involved treating beer with either hop extracts or

268 isinglass at 12°C and maintaining the sample at that temperature, to mimic a cask ale

269 process. The haze was then measured at 12°C, 72h after treatment, to determine the

270 maximum amount of haze that could be formed under these conditions. Relative to the

271 unfined control, all treatments reduced the level of haze in the beer sample (Figure 5). The

272 lowest hop extract treatment resulted in the lowest level of haze, with haze increasing with

273 increasing doses of the extract. However, even when added at 8 times the optimal dose-rate

274 the level of haze was not as high as that observed for the unfined sample.

275 The second protocol was designed to mimic a lager beer application. In these experiments

276 the finings were applied at 4°C and the samples maintained at 4°C thereafter. The samples

277 were analysed unfiltered (Table 1), and results showed that both isinglass and the hop

278 extract had substantially reduced the level of haze in the sample. The levels of haze were

279 greater for the samples with the hop extract, relative to those fined with isinglass and as

280 with the cask style experiment the level of haze increased at doses of the hop extract in

281 excess of the optimum. It was however clear from the data, that the levels of haze obtained

282 with the lowest doses of isinglass and hop extract were only marginally different.

283 To further evaluate the extracts under lager-style process conditions the beers were filtered

284 sequentially. The unfined control beer was typically more hazy than the fined samples

285 throughout the filtration process. Following the 0.45µm filtration step, there were few real

286 differences between the fining treatments, with substantial reductions in haze for both

287 isinglass and the hop extract.

288 4. Discussion

289 Proanthocyanidins have been reported in hops, but, typically the size is significantly smaller

290 than those found in the current study. Proanthocyanidins reported by Li and Deinzer (18)

291 contain only a few polymer subunits. Those reported by Taylor et al. (19) were larger

292 proanthocyanidins with up to 20 sub-units (average 7.8) which would equate to a molecular

293 weight of around 6000Da. These are still significantly smaller than the proanthocyanidin

294 molecular weight ranges suggested by our molecular weight fractionation studies, which

295 imply the presence of polymers 10 times that size, or, even larger.

296 Rodrigues et al. (20) reported that proanthocyanidins are readily absorbed onto yeast lees

297 in wine. The size of the polymers observed in the hop extracts appear to be sufficiently large

298 to stick to not just one yeast cell but to join cells together and flocculate them. This results

299 in the observed fining activity. Proanthocyanidins are also known to act as antioxidants,

300 chelate metal ions and bind with proteins (3; 15; 21), further activities that may be

301 beneficial as brewing processing aids and would also be consistent with the reduction in

302 haze observed during the fining experiments. Considering the likely proanthocyanidin

303 nature of the active material, it is also apparent that over-addition of the finings has the

304 potential to induce haze in samples. This will depend on the levels of haze-sensitive proteins

305 present, and thus on the stabilisation regime a beer has been subjected to. In this particular

306 example (Figure 5), it was possible to dose the extract at up to 8 times the determined

307 optimal dose, without increasing haze relative to the unfined control. Thus, by adopting

308 customary procedures for optimising the dose rates of finings the potential negative

309 consequenses of over-dosing would easily be avoided.

310 The use of polyphenol-rich extracts in the brewhouse has been reported as one potential

311 route to improve the colloidal and flavour stability of beers (22; 23). Jelinek et al. (22)

312 reported a reduction in haze-active prolamines in beer when brewing (kettle addition) with

313 the addition of residual material from the processing of Saaz hops into T45 pellets. This

314 fraction contained almost 10% w/w total polyphenols. However, the possibility of using such

315 spent residues as a source of fining activity has not previously been reported, presumably

316 because addition in the brewhouse results in the degradation and removal (as trub) of large

317 polymeric proanthocyanidins.

318 Experiments using successive filtration post-fining (data in Table 5) were designed to

319 evaluate whether an aqueous extract of spent hops could match the performance of

320 isinglass in terms of haze reduction in a lager-type application (where beers would typically

321 be filtered post-fining). We have identified that acetone extracts of spent hops match and

322 even exceed isinglass in this regard (24). However, aqueous conditions appear to extract a

323 wider range of material from spent hops and are more prone to inducing additional haze if

324 not used at the optimal dose rate. Here it was shown that filtration post-fining enabled the

325 aqueous hop extract to broadly match the performance of isinglass in terms of total haze of

326 the filtered beers at 4°C.

327 Many of the attributes of the hop extracts make them suitable for use in the brewing

328 industry. The sediments formed following fining action are compact and not fluffy, the

329 sedimentation rate is fast (hence the dose sediment curve in Figure 1 was determined 2h

330 after extract addition) and they are of plant origin without need of chemical modification.

331 The results presented in this paper were obtained with extracts derived from the hop

332 variety Galena. However, hop extracts of other varieties have also been shown to be active

333 flocculants (24). The original activity was observed during dry hopping experiments where

334 the levels of hop addition were within typical brewing ranges. The use of a hop extract,

335 allows for an efficient use of previously extracted material, at less than 0.5g of CO2-

336 extracted spent hops per litre. This may ultimately enable a wider use of these extracts

337 within the brewing industry.

338

339 References

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Table 1: Total haze (90° scatter) at 4°C for beers treated with isinglass or aqueous hop extract (fold dosage relative to optimum) and sequentially filtered.

Treatment Unfiltered 11µm filter 3µm filter 0.45µm filter

Unfined 15.06 9.48 1.80 1.04

0.5x Isinglass 2.86 1.85 1.09 0.27

1x Isinglass 1.65 1.04 0.71 0.20

0.5x Hop Extract 3.11 2.88 1.15 0.22

1x Hop Extract 3.77 2.95 1.12 0.89

4x Hop extract 4.50 3.70 2.15 0.23

1.4

1.2

0.8

0.6

OD 600nm (Au) 600nm OD 0.4

0.2

0 0 2 4 6 8 10 Hop extract addition rate (% v/v)

Figure 1: OD 600nm of green beer 2h after treatment with varying amounts of hop extract at 4°C.

25 IG 4 HE 4

20 IG 22 HE 22

10 Sediment volume (ml/L) volume Sediment 5

0 0.5 1 2 4 Dosage Rate (ratio relative to optimum)

Figure 2: Sediment volumes (ml/L of beer) formed by the addition of varying amounts of isinglass

(IG), or hop extract (HE) to green beer at 4°C (solid markers), or 22°C (open markers).

1.30

1.00

0.70 Absorbance (Au) 0.40

0.10 450 500 550 600 Wavelength (nm)

Figure 3: Visible absorbance spectrum of the butanol/HCl-hydrolysed hop extract

287 100

313 257 271 303 343 0 m/z 260 280 300 320 340

Figure 4: Mass spectrum of the butanol/HCl-hydrolysed hop extract.

2.5

1.5

1 Haze (H90) Haze

0.5

0 Unfined IG 0.5xHE 1xHE 4xHE 8xHE Treatment

Figure 5: Total haze (90° scatter) at 12°C for green beer treated with isinglass (IG), varying amount of

aqueous hop extract (HE, fold dosage relative to optimum), or unfined 72h after treatment.